Abstract Our past research has provided considerable insight into the signals elicited through G-protein coupled receptors (GPCRs) that activate RhoA on astrocytes and in 1321N1 glioblastoma cells. The pathways regulated through PAR1 and S1P receptors via G?12/13 engagement include RhoA activation to increase cell proliferation, survival, and invasion-hallmarks of cancer. Our recent studies demonstrate robust activation of the transcriptional co-activators MRTF-A and YAP through RhoA signaling in glioblastoma cells, and implicate altered gene expression in proliferative and migratory responses. The objective of this proposal is to demonstrate that robust transcriptional gene programs elicited through RhoA signaling are critical to glioblastoma multiforme (GBM) tumor growth and maintenance of glioblastoma stem cells (GSC), with a long term goal of identifying new therapeutic targets for this devastating disease. Aim #1 uses the human 1321N1 glioblastoma cell line to define cellular events and identify changes in specific target genes by which GPCRs and RhoA engage transcriptional pathways that contribute to cancer-relevant cellular responses. S1P- and thrombin-induced proliferation, survival, adhesion, migration, invasion and angiogenesis are assessed in WT and MRTF-A or YAP CRISPR/Cas9 KO cells. Data from RNA seq analysis are used to identify critical regulated genes, and tested for their functional importance in cellular responses. Actions of target genes on, autocrine and transcriptional pathways that amplify response to GPCRs and RhoA are considered. Aim #2 uses patient-derived glioblastoma xenografts (PDX), as a model of glioblastoma stem cells. Several PDX lines will be grown in serum free medium in vitro as neurospheres or adherent cultures and subsequently implanted as orthotopic (brain) xenografts... YAP, MRTF-A, RhoA and their downstream target genes will be knocked down using shRNA and in vitro stem cell markers, cellular responses and in vivo tumor growth assessed. Aim #3 tests the hypothesis that RhoA-mediated transcriptional signaling leads to astrocyte dedifferentiation and gliomagenesis driven by activated Ras). Proof of principle experiments examine dedifferentiation of isolated mouse astrocytes infected with lentiviruses encoding oncogenic Ras and in which molecules in the RhoA signaling pathway are genetically deleted or knocked down with shRNAs. In vivo studies of gliomagenesis are carried out by delivery of oncogenes by lentiviral infection into the hippocampus of GFAP-Cre mice followed by analysis of tumor growth, invasion, and changes in expression of target genes and stem cell markers. The overall findings from these studies should demonstrate that GPCR- and RhoA-mediated transcriptional activation can elicit genetic and functional responses that contribute to dysregulation in glioblastoma, and that RhoA signaling contributes to the oncogenic effect of established GBM tumor drivers. The health-related significance is that these findings could shift the focus of current research and clinical practice from the established disease drivers towards consideration of GPCR- and RhoA-regulated signaling pathways in GBM.